Fig 1.
Sampling regimes of Buergeria japonica sensu lato in this study.
(A) Sample localities, sample sizes, and genetic assignments from molecular evidence; (B) sample localities, sample sizes, and call types from acoustic evidence; and (C) the fine-scale sampling regime in Taiwan. Populations from Ryukyu and northwestern Taiwan (Japanese clade) could express only one type of long calls (Type 1A in light blue), but those from eastern and southern Taiwan (Taiwanese clade) are capable to express two: one very similar to the former (Type 1B in dark blue), and the other exclusively occurring in this region (Type 2 in orange). Arabic numerals in the pie charts indicate samples sizes for molecular analysis; while percentages in column charts indicate the population ratio in each drainage which was recorded to express either types of the calls.
Table 1.
Region, locality, abbreviation, sample sizes (acoustic, morphological, molecular, and number of mitochondrial haplotypes), and GenBank accession numbers for each Buergeria japonica population used in this study.
Fig 2.
Maximum likelihood phylogeny of Buergeria japonica sensu lato.
Phylogenetic tree was constructed by using PhyML 3.0 based on complete cytochrome b sequences, with 1000 bootstrap replicates and Bayesian posterior probability as statistic supports. Japanese clade (blue) and Taiwanese clade (orange) represent 16.19% between-group sequence divergence (p-distance).
Table 2.
Within-clade (diagonal) and between-clade (upper-right) divergences among the four major lineages in the Japanese clade (Buergeria japonica) and two major lineages in the Taiwanese clade (Buergeria otai sp. nov.).
Fig 3.
Comparison of long call sonograms between the Japanese and Taiwanese clades.
The Japanese clade (Buergeria japonica) could express only one type of long call (Type 1A), yet the Taiwanese clade (Buergeria otai sp. nov.) is capable to express an additional type (Type 1B and Type 2). The units (u or ku) displayed on the vertical axis of a waveform view are the actual sample values in the signal, which are proportional to the sound pressure at the microphone when the sound was recorded (Raven Pro v1.4, Cornell Lab of Ornithology, Ithaca, NY, USA). Sonograms in this figure: Type 1A: No. 150518_03#2 from Wu Stream, northwestern Taiwan; Type 1B: NMNS 19824 (paratype of B. otai sp. nov.) from Donggang Stream, southern Taiwan; Type 2: NMNS 19816 (paratype of B. otai sp. nov.) from Donggang Stream, southern Taiwan.
Fig 4.
Comparison of short call sonograms between the Japanese and Taiwanese clades.
The Japanese clade (Buergeria japonica, abbreviated as JP) expresses calls with a comparatively slower tempo, which was represented by their significantly higher call duration (DT, in sec; F1,13 = 20.75, p<0.001) and call rise time (RT, in sec; F1,13 = 20.26, p<0.001). On the other hand, the Taiwanese clade (B. otai sp. nov., abbreviated as OT) expresses a lower dominant frequency (DF, in kHz; F1,13 = 5.59, p = 0.03). Call fall time (FT) and interquartile range (IQR) were not significantly different the two clades. The units (u or ku) displayed on the vertical axis of a waveform view are the actual sample values in the signal, which are proportional to the sound pressure at the microphone when the sound was recorded (Raven Pro v1.4, Cornell Lab of Ornithology, Ithaca, NY, USA). Sonograms in this figure: B. japonica: No. 150518_03#2 from Wu Stream, northwestern Taiwan; B. otai sp. nov.: NMNS 19816 (paratype) from Donggang Stream, southern Taiwan.
Fig 5.
Principal component analysis and morphological differentiation between the Japanese and the Taiwanese clades.
The Japanese clade (Buergeria japonica) is abbreviated as JP and represented in blue; and the Taiwanese clade (Buergeria otai sp. nov.) is abbreviated as OT and represented in orange. These two clades differed significantly in PC2, which was consisted majorly by the size and shape of the head (F1,14 = 15.85, p = 0.001).
Fig 6.
Buergeria japonica (A, C) and Buergeria otai sp. nov. (B, D, E, F) in live.
The irregular patches of B. japonica on the thighs could be compared to the regular tiny spots of Buergeria otai sp. nov., regardless the intraspecific variation from very few (E) to many (F). Photographed by YJ Liang (A, C), CM Tsao (B), and HN Nguyen (D, E, F).
Fig 7.
Behavioral response of male frogs toward conspecific and heterospecific calls.
Both species expressed higher defensiveness against conspecific rather than heterospecific calls. Buergeria japonica expresses highest “Response call numbers” (A) and “Response call ratio” (B) toward Type 1A long call; while Buergeria otai sp. nov. expresses highest response to Type 2 (D and E). Conclusively, both species choose the conspecific call as their “Highest defense opponent” (C and F). It is noteworthy that B. japonica is not able to distinguish between Type 1A (conspecific) and Type 1B (heterospecific) owing to the similarity.
Fig 8.
Holotype (A and C, male, NMNS 19819) and one of the paratypes (B and D, female, NMNS 19871) of Buergeria otai sp. nov.